Spectroscopic determination of characteristic of biological material

ABSTRACT

Method for determining a cellular function or change in cellular function by contacting a sample of cells or a component of cells with an activating agent, directing a beam of infrared light at the sample of cells or a component of the cells, analyzing the infrared spectrum of the sample at at least one range of frequencies and ascertaining whether at least one change in the spectral characteristics has occurred due to activation of the cells or a change in the cellular component, that can be correlated to the cellular function or the change in cellular function by the activating agent and determining the cellular function therefrom.

The present invention relates to a spectroscopic method for theinvestigation of biological material. The invention is particularlyconcerned with the application of Infrared (IR) spectroscopy(spectrometry) in the investigation of blood or other body fluids or oneor more components thereof.

An example of an investigation in accordance with the invention is thedetermination of cellular immunity in patients with immunodeficiency,autoimmunity, contact with infectious diseases, allergies,hypersensitivities, and cancer. The investigation may be related todetermining tissue compatibility for transplants.

BACKGROUND OF THE INVENTION

Currently there are a number of clinical laboratory methods ofdetermining cellular immunity. The delayed hypersensitivity skin test isone tool that occasionally serves to establish a diagnosis in areas suchas allergy studies. However patients who are highly sensitive to variousantigens will have marked reactions to such skin tests. In some casesskin tests cannot be performed at all to avoid challenging the patientwith potentially hazardous antigens.

Another technique for determining cellular immunity is lymphocyteactivation. Lymphocyte activation, also known as lymphocyte stimulation,refers to an in vitro correlate of an in vivo process that generallyoccurs when antigen reacts or interacts with specifically sensitizedlymphocytes in the host. The lymphocyte activation or stimulation testis one in which lymphocytes are extracted from whole blood and incubatedwith an antigen. Tritiated thymidine is then added over a 16 hour periodbefore the cells are harvested and their radioactivity measured by useof a liquid scintillation counter. This in vitro technique may be usedto assess cellular immunity in patients with immunodeficiency,autoimmunity, infectious diseases, allergies or hypersensitivities andcancer and in the area of transplantation compatibility.

The disadvantages of existing in vitro assays based on lymphocytes andmonocytes is that they are time consuming, labour intensive, impreciseand expensive because of the reagents, equipment and highly qualifiedlabour required.

SUMMARY OF THE INVENTION

I have found surprisingly that IR spectroscopy may be used toinvestigate blood or other body fluid or component(s) thereof. The useof IR spectroscopy has the advantages of providing results relativelyquickly with less labour input when compared to the conventionalmethodology. Moreover the use of IR spectroscopy may provide a moreprecise indication of a characteristic of a blood or body fluidcomponent. A further advantage of the use of IR spectroscopy is that itallows the identification of dynamic processes through changes in the IRspectra.

The term body fluid components include sweat, saliva, urine, semen andlacrimal secretions.

IR spectroscopy is routinely used by organic chemists and biochemistsand others as a molecular probe. When infrared light is passed through asample of an organic compound, some of the frequencies are absorbedwhile other frequencies are transmitted through the sample without beingabsorbed. By IR spectroscopy we also include Laser-Raman spectroscopyincluding Raman confocal laser spectroscopy or any other IR spectroscopytechnique.

Organic applications of IR spectroscopy are almost entirely concernedwith frequencies in the range 650-4000 cm⁻¹. Frequencies lower than 650cm⁻¹ are called far infrared and those greater than 4000 cm⁻¹ are callednear infrared.

Conventional IR spectrometers suffer disadvantages in sensitivity, speedand wavelength accuracy. Most spectrometers scan over the wavelengthrange and disperse infrared light using a grating or prism. Thesedispersive infrared spectrometers suffer from wavelength inaccuraciesassociated with backlash in the mechanical movement, such as rotation ofmirrors and gratings.

An entirely different principle is involved in Fourier Transforminfrared (FTIR) spectroscopy , which centres on a Michelsoninterferometer. The FTIR spectrometer has the advantage of speed andsensitivity in which picogram quantities of sample can give goodspectra.

The present invention provides, in one aspect, a method for theinvestigation of:

at least one component of blood or other body fluid;

the change(s) in the at least one component;

the functional status of the at least one component; or

the change in the functional status of the at least one functionalcomponent

the method including directing infrared light through a samplecontaining the at least one component and analysing the absorptioncharacteristics of said sample.

Preferably the method of the invention is carried out using FTIRspectroscopy but other IR spectroscopic techniques may be used.

The absorption characteristic determined may be those in the region ofsymmetric and antisymmetric stretching modes of phosphodiester groups,the C—O stretching mode, the CH₂ bending mode, and the amide I and IIbands. The absorption characteristics analysed may be those due tofunctional group vibration in signature molecules or groups, forexample, the phosphodiester group of nucleic acids, COH groups, C—Ogroups of, for example, fatty acyl groups or glycogen bands,carbohydrates or due to lipid molecules present in the specimen.

The reference to blood and body fluid components may include, but is notlimited to, single or mixed cell populations, a single simplebiochemical component or complex mixtures of biochemical componentsderived or prepared from blood or body fluids.

The investigation may be carried out on whole blood or other body fluidor an extract of component thereof. The component may be, for example,lymphocytes, erythrocytes or platelets.

The method of the present invention has particular application in thedetermination of cellular function or change in cellular function.

Accordingly, in a further aspect, the present invention provides amethod for determining a cellular function or change in cellularfunction of cells, the method including:

contacting a sample of the cells or a component of the cells with anactivating agent;

directing a beam of infrared light at the sample of a cells;

analysing the infrared absorption of the sample at at least one range offrequencies; and

ascertaining whether at least one charge in the absorptioncharacteristic has occurred due to activation of the cells by theactivation agent and determining the cellular function or change incellular function therefrom or correlating the change in component ofthe cell to a change in the cellular function.

The cellular function determined may be any function that is anindicator of viability, integrity or functional status of the cells. Thefunctional status may be immune competence.

The cells used in the method of the invention may be selected fromlymphocytes or erythrocytes. Preferably the cells are lymphocytes.

Lymphocytes may be isolated by purification of anticoagulated peripheralblood by any suitable technique, for example density gradientcentrifugation or use of magnetic beads.

The activating agent may be a biological or non-biological agent(s).These agents may be naturally derived or synthetic. Examples of thesebiological or non-biological agents include, but not limited to:

a) Mitogens which are non-specific agents which stimulate or activatelarge numbers of lymphocytes and do not require a sensitized host.Mitogens cause a myriad of biochemical events and ultimately division oflymphocytes. Examples of mitogens include concanavilin A,phytohaemagglutinin, Staphylococcus Protein A, pokeweed mitogen, phorbolmystirate acetate and Streptolysin S.

b) Potential antigens or previously encountered antigens which have asensitized host and stimulate specific cells, in most instances, T or Blymphocytes or other immunocompetent cells that are or becomespecifically sensitized to the antigens in question. Antigens mayinclude but not be limited to:

i) live, attenuated or dead microorganisms or components or productsfrom microorganisms whether naturally occurring, synthetic orgenetically engineered such as cell surface lipopolysaccharide ortoxins, for example, Candida antigen, Streptokinase, tetanus toxoid,vaccinia virus, and Herpes simplex virus;

ii) cells or cellular components or products derived from plants,animals, whether naturally occurring, synthetically induced, geneticallyengineered including cell surface components. Included in this categoryare antigens, either presented on cells or isolated from cells such asHistocompatibility antigens, ABO blood group antigens, virally inducedcell components or surface markers, cell developmental ordifferentiation markers, tumour-induced or tumour-specific components,and haptens or moieties whose binding to cell subsequently induces thecells to become stimulated or activated or whose binding to isolatedcell components causes an alteration which can be correlated to cellstimulation or activation.

c) Monoclonal or polyclonal antibodies to lymphocyte cell surfacemolecules which can result in activation or cell death.

I have found that the dynamic cellular processes which are known tooccur in lymphocyte activation/stimulation are manifest as changes overtime in the infrared spectral profile of the activated lymphocytescompared to unactivated lymphocytes. The determination of the inventionmay be carried out by measuring the IR spectral profile of the sampleand comparing that with the “normal” or alternatively by investigatingthe change of the spectral profile over time.

The IR spectral profile may be determined two or more times over aperiod and the spectral profiles compared to determine whether at leastone change in an absorption characteristic in one or more regions of theprofiles has taken place. I have found that in some instances changes inthe spectral profile may take place within 30 minutes.

Alternatively, the determination may be carried out by taking aninfrared spectrum of a sample and comparing it with a standard spectrumand ascertaining whether there is at least one differences in anabsorption characteristic in one or more regions of the profiles.

In yet another aspect the present invention provides a method for thedetermination of the immunocompetence and/or disease status of a humanor animal subject, the method including taking a sample of blood orother body fluid from the subject and subjecting the sample or anextract therefrom, optionally after being contacted with a stimulatingagent, to infrared radiation to provide an IR spectral profile thereofand determining the lymphocyte function and/or activation as a measureof the immunocompetence and/or disease status of the subject.

The present invention may also be used to investigate the viability andfunctional integrity of blood or a blood component such as erythrocytesor platelets over time and conditions of storage. This can be achievedby comparing the IR spectra profile with the profile of “fresh” materialand determining whether there are any differences in the spectra. Thishas particular application in blood banks and the like where the presentinvention provides a relatively speedy method of determining theviability and functional integrity of stored blood.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a series of spectral profiles taken at intervals from 30seconds through to 180 minutes.

EXAMPLES

In order to assist in an understanding of the present invention weprovide the following non-limiting examples.

Example 1

FTIR Monitoring of Lymphocyte Activation

Peripheral blood mononuclear cells (PBMC) from two volunteers, isolatedusing gradient mixtures were twice washed in 0.9% saline andcentrifuged. The resulting pellets were resuspended in 1 ml of saline.One half of each sample was activated with phorbol myristate acetate(PMA) and the other half used as a control. After 15 minutes the fourportions were desiccated and transferred to a infrared cell for FTIRmicroanalysis. For each portion six spectra were recorded.

A high quality, highly reproducible spectra of activated andnon-activated lymphocytes from both volunteers resulted. Spectra of theactivated lymphocytes exhibit marked differences to those of thenon-activated lymphocytes. Spectra of activated lymphocytes arecharacterised by a reduction in the α-helical amide I band and anincrease in the band associated with amide Iβ-pleated sheet component at1634 cm-⁻¹. The amide II band exhibits a multitude of shoulder peaksindicative of β-turns associated with protein conformational changes.The C—O stretch of fatty acyl groups (1400 cm⁻¹) and the C—O stretchesof glycogen bands at 1058 cm⁻¹ and 1038 cm⁻¹ are much more intense inactivated lymphocytes. The band at 1295 cm⁻¹ appears to be shifted from1286 cm-⁻¹ in the non-activated activated cells.

The results of these PMA experiments demonstrate the potential of FTIRspectroscopy to not only detect the initial allogeneic activation oflymphocytes but also provide a wealth of molecular information connectedwith the triggering of the immune response.

Example 2

Blood from two HLA disparate volunteers (L and P) was diluted in 0.9%saline. 10 ml of Lymphoprep™ was then carefully layered beneath thediluted blood and the tubes centrifuged at 2300 g for 15 min. Theresulting lymphocyte layers were separately transferred, twice washedand centrifuged and finally resuspended in 2 ml of isotonic saline.Aliquots were collected from each tube and mixed in nine Eppendorf tubesand a further nine from P and nine from L were placed in separateEppendorf tubes to serve as controls. The lymphocytes were centrifugedat 2600 g for 5 minutes and placed in a 37° incubator. At times between0 and 180 min., 100 μL volumes from the mixed, L and P tubes weretransferred into the wells of a infrared cell and then rapidlydesiccated. The resultant thin pellets were analysed with the FTIRmicroscope. Blood from the two HLA identical twins was used to obtain IRspectra following exactly the same methodology. Blood from 2 individualswith a 50% HLA disparity was worked up in the same manner but withtissue culture growth medium substituted for the isotonic saline as theincubation medium.

After 5 minutes (see FIG. 1) no apparent changes in the spectralprofiles were evident. At 15 minutes there appears to be an increase inphosphate bands at 1238 cm⁻¹ and 1086 cm⁻¹ and after 30 minutes radicalspectral changes are observed. The amide II band is reduced and shoulderpeaks indicative of P turns associated with protein conformationalchanges have become more pronounced. The band at 1393 cm⁻¹ from the C—Ostretch of fatty acyl groups has dramatically increased and a sharp bandat 1286 cm⁻¹ has appeared. The PMA activated lymphocytes producedspectra with a similar band at 1295 cm⁻¹. The most striking features areobserved in the carbohydrate/phosphodiester region (1200-1000 cm⁻¹),with a dramatic increase in bands associated with the C—O stretches ofcarbohydrates at 1004 cm⁻¹ and 1058 cm⁻¹. This feature was also observedin the spectra of lymphocytes activated with PMA and may reflect anincrease in surface glycoproteins. After 60 minutes the spectral profilestill resembles the 30 minute profile, however in the later samplesthese changes have dramatically resided indicating a quiescent period.

For the 50% HLA disparate individuals spectral changes almost identicalto those above occurred but after a much longer time delay (55 min.).These changes persisted for similar length as those in the spectra ofthe HLA disparate individuals. This result implies that the allegenaicstimulation spectra of individuals who share some HLA alleles exhibitspectral changes suggestive of activation at longer time intervals. Thisimplication is supported also through the use of tissue culture mediuminstead of saline for the incubation medium which in theory shouldenhance lymphocyte activation times.

The time series infrared spectra of the allegenaic stimulatedlymphocytes from HLA identical siblings exhibit none of the changessuggestive of lymphocyte activation over the whole 180 min. time periodsupporting the hypothesis that the allegenaic stimulated infraredspectra of individuals with compatible immunologic alleles take a longerincubation time period to exhibit spectral changes suggestive ofactivation. In the case of the HLA identical twins we would not expectto observe such changes, however only a longer time series study willconfirm this hypothesis. These preliminary results demonstrate thepotential of IR spectroscopy to revolutionise matching protocols in thearea of tissue transplantation. The chemical information available fromthe infrared spectra should also help clarify the biochemistry ofactivation.

The claims defining the invention are as follows:
 1. A method fordetermining a cellular function or a change in cellular function, themethod including: contacting a sample of cells with an activating agent;directing a beam of infrared light at the sample of cells; analyzing theinfrared spectrum of the sample at at least one range of frequencies;and ascertaining whether at least one change in the infrared spectrumhas occurred due to the activation of the cells that can be correlatedto the cellular function or a change in cellular function by theactivating agent and determining the cellular function or the change incellular function therefrom, wherein the sample is a body fluidcontaining said cells.
 2. A method according to claim 1, wherein thesample is blood or a component thereof containing at least one cellularcomponent selected from the group consisting of lymphocytes,erythrocytes and platelets.
 3. A method according to claim 1, whereinthe cells are immunocompetent cells sensitized to an antigen.
 4. Amethod according to claim 3, wherein the immunocompetent cells arelymphocytes.
 5. A method according to claim 1, wherein the infraredlight is produced by a Fourier Transform Infrared spectrometer.
 6. Amethod according to claim 1, wherein the infrared light is produced by aRaman confocal spectrometer.
 7. A method according to claim 1, whereinthe activating agent is one or more mitogens.
 8. A method according toclaim 1, wherein the activating agent is one or more antigens.
 9. Amethod according to claim 1, wherein the activating agent is selectedfrom the group consisting of a monoclonal antibody, polyclonal antibodyand a ligand to a cell component.
 10. A method according to claim 1,wherein the cellular function or change in cellular function determinedis cellular immunocompetence.
 11. A method according to claim 10,wherein the cellular function or change in cellular function determinedis cellular immunocompetence and wherein the sample is taken from asubject with immunodeficiency, autoimmunity, potential contact withinfection disease, allergies, hypersensitivy or cancer.
 12. A methodaccording to claim 1, wherein the cellular function or a change incellular function determined is tissue compatibility for transplants.13. A method according to claim 1, when the method is used to determinetissue compatibility for tissue or organ transplant.
 14. A methodaccording to claim 1, wherein the analysis of the infrared spectrumincludes analysis of spectral characteristics of at least one range offrequencies to ascertain whether (a) at least one change in the infraredspectral characteristics has occurred due to vibration of at least onefunctional group of molecules, (b) at least one change in the infraredspectral characteristics has occurred due to conformational changes ofat least one functional group of molecules, or both (a) and (b) in thesample.
 15. A method according to claim 14, wherein the at least onechange in the infrared spectra characteristic is a change in absorptionintensity at a particular frequency or a change of frequency at which aparticular absorption occurs.
 16. A method according to have claim 14,wherein the at least one functional group is in at least one moleculeselected from the group consisting of carbohydrates, nucleic acids,lipid molecules, proteins, glycoprotein, and glycogen.
 17. A methodaccording to claim 14, wherein the at least one functional group isselected from the group consisting of a phosphodiester group, a C—OHgroup, a CH group and CH₃.
 18. A method according to a claim 1, whereinthe analysis of the infrared spectrum of the sample is carried out twoor more times.
 19. A method according to claim 1, wherein the at leastone range of frequencies is in the range of 950 cm⁻¹ to 1650 cm⁻¹.